1. Field of the Invention
The present invention relates to a cucurbituril derivative-bonded solid substrate, and more particularly, to a solid substrate covalently bonded with a cucurbituril derivative which can immobilize a biomaterial by a non-covalent interaction, and its applications.
2. Description of the Related Art
After the human genome sequence was drafted in 2000, gene expression could be understood at mRNA level. Thereafter, developments of personalized medicines or diagnostic reagents based on individual genome information have been anticipated. Therefore, there has arisen a need to rapidly trace the expression levels of a large number of genes. In this regard, there was developed a DNA chip capable of simultaneously performing assays of a thousand to ten thousand genes. However, a gene assay alone cannot provide information about proteins which are not only gene products but also biomaterials essential for biological activity. Therefore, there has been suggested a protein chip, a corresponding concept of a DNA chip, which can perform simultaneous assays of a large number of proteins.
The concept of the protein chip is based on the protein microarrays which contain chemically or biochemically treated surfaces for specific interaction with proteins of interest. A protein chip can be made by using a solid substrate illustrated in FIG. 1 as follows: first, a thin film is formed on a solid substrate 4 using compounds with functional groups 1 for linkages with the solid substrate 4 and another functional groups 3 for linkages with biomaterials such as proteins. Then, biomaterials such as proteins can be immobilized on the solid substrate 4 via chemical or physical interactions between biomaterial and the terminal functional group 3. In FIG. 1, 2 refers to a molecular body.
Hitherto, many researchers have used covalent bonds between the functional groups 3 of Reference Diagram 1 and proteins to immobilize the proteins on a solid substrate. When covalent bonds between the functional groups 3 of Reference Diagram 1 and proteins are formed, the proteins can be immobilized on the surface of a solid substrate.
However, it is well-known that their specificities or activities toward substrates are seriously affected by the immobilization method, because the specificity and activity are strongly related to their specific three-dimensional structures and orientation of their active site. Therefore, the three-dimensional structures of proteins may be damaged when the proteins are covalently bonded to a solid substrate, thereby causing degeneration of the proteins, such as a protein 6 shown in FIG. 2. This is because the function of proteins is dependent on their specific three-dimensional structures formed by chains of amino acids constituting the proteins. To maintain the function of a protein chip, like a protein 7 in FIG. 2, an active site must not be bonded to the linkage layer 5 to preserve the functionality of the active site.
To solve this problem, many methods have been developed for immobilizing proteins to a surface of a solid substrate via non-covalent bonds.
By way of an example, a study about the attachment of proteins to a solid substrate by a coordination bond was reported. Paborsky et al. suggested a coordination linkage between proteins fused with histidine, which is an amino acid known to well bind with Ni, Cu, etc., and a surface of a solid substrate to which Ni is attached by nitrilotriacetic acid (NTA) [Paborsky, L. R.; Dunn, K. E.; Gibbs, C. S.; and Dougherty, J. P., Anal. Biochem. 1996, 234, pp. 60-65].
Frey et al. reported the attachment of an intermediate, such as polylysine, capable of ionically binding with proteins, to a solid substrate, to immobilize the proteins on the solid substrate [Frey, Brian L.; Jordan, Claire E.; Kornguth, Steven; and Corn, Robert M., Anal. Chem. 1995, 67, 4452-4457].
Recently, Tae-Sun Kim et al. reported a hydrogen bond between proteins and a solid substrate having crown ether derivatives, paying attention to a hydrogen bond between ammonium groups abundantly present at non-active sites of proteins and crown ether groups (Korean Patent Application Nos. 10-1999-0061074 and 10-2000-0038491).
However, the bond strength of most non-covalent bonds is much less than that of covalent bonds. Therefore, proteins having non-covalent bonds with a solid substrate may be detached from the solid substrate when contact with chemical materials used in immunoassay. In this regard, many attempts have been made to immobilize proteins on a solid substrate via stronger non-covalent bonds.
Recently, Yao and co-workers reported a solid substrate for a protein chip in which avidin, a type of protein, is immobilized on the solid substrate via a covalent bond [Lesaicherre, M.-L.; Lue, R. Y. P.; Chen, G. Y. J.; Zhu, Q.; and Yao, S. Q. J., Am. Chem. Soc. 2002, 124, 8768]. Avidin is known to bind with four small molecules of biotin by a coupling constant of about 1015 M−1, which is the strongest non-covalent bond among currently known non-covalent bonds [Wilchek, M.; Bayer, E. A. Avidin-Biotin Technology. In Methods in Enzymology 1990, 184]. According to the report by Yao et al., probe proteins are fused with biotin and then are immobilized on a solid substrate treated with avidin. Reportedly, the probe proteins are not detached from the solid substrate even under a very severe environment. However, this method has an economical limitation of avidin being costly, even though there is an advantage in that a coupling constant of avidin-biotin interaction is very large.
Therefore, a cost effective method for immobilizing proteins to a solid substrate using a non-covalent bond with strong interaction is required.